A Molecular Model of Phosphorylation-Based Activation and Potentiation of Tarantula Muscle Thick Filaments

Abstract

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non- and 50% mono-phosphorylated, while on activation mono-phosphorylation increases and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35 while Ca2+-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest they are the targets for protein kinase C (PKC) and myosin light chain kinase (MLCK) respectively. The atomic model of the tarantula filament shows that the two myosin heads (“free” and “blocked”) are in different environments, with only the free head serines readily accessible to kinases. Thus PKC Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest these heads are less strongly bound to the filament backbone, and may oscillate occasionally between attached and detached states (“swaying” heads). These heads would be available for immediate actin interaction upon Ca2+-activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked-head Ser45 to MLCK. This would release the blocked-heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca2+ on tetanus would recruit new, MLCK-activated heads, resulting in force potentiation.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

“…However achieving the near-atomic resolution IHM structure on frozen-hydrated thick filaments seems to be a difficult task on view that only~2-nm resolution has been achieved on the heads region of the 0.55-nm near-atomic resolution Lethocerus 3D map . This is probably a consequence of the intrinsic flexibility of the IHM paired heads arrangement (Yang et al 2015a) especially of the free swaying heads as explained below (Brito et al 2011;Sulbarán et al 2013). Interestingly, it has been recently reported that a lack of negative charge in S2 may explain why Lethocerus thick filaments display an IHM perpendicular to the filament axis, rather than a parallel one as seen in tarantula (Fee et al 2017).…”

“…The earlier tarantula IHM 3DTP quasi-atomic model IHM structure imposes different heads environments explaining heads ordered release on activation The 3DTP model has both IHM heads asymmetrically arranged; so their RLC phosphorylatable serines, Ser35 and Ser45, were not similarly exposed to its specific kinases, protein kinase C (PKC) and myosin light chain kinase (MLCK) (Alamo et al 2008;Brito et al 2011) (Fig. 7a).…”

“…7a). (Brito et al 2011;Sulbarán et al 2013). In the relaxed state, the blocked head is immobilized as it is docked onto its S2 and neighbor myosin tail, whereas the free head is docked to the blocked head and allowed to sway away (Fig.…”

“…So the working in cooperation of the two heads in myosin II working in cooperation is crucial to allow these specialized physiological responses of skeletal muscle to sustain relaxation and control the force produced during contraction. This model for activation, potentiation and postetanic potentiation (Brito et al 2011;Sulbarán et al 2013) explains its cooperativity so it was called the cooperative phosphorylation-activation (CPA) mechanism (Sulbarán et al 2013). …”

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

“…Phosphorylation of the analogous serine in smooth muscle RLC (smRLC) is the primary mechanism of contractile regulation in that tissue. Phosphorylation of skRLC plays a similar role in some invertebrate skeletal muscles (23,24) and enhances contractility in mammalian skeletal muscle (25).…”